北京地区核桃黑斑病病原菌的分离、致病性测定和16S rDNA序列分析

Identification and pathogenicity assay of the pathogen of walnut blight in Beijing

核桃黑斑病是危害核桃生产的重要细菌病害,造成树势衰弱,减产严重,甚至树体死亡。2008年在北京郊区栽植的幼树上发现疑似病害,且呈爆发之势。为确定其病原菌分类地位,用组织分离法分离核桃黑斑病菌,纯化培养后用针刺接种法回接到健康核桃树枝条和叶片上,14 d后叶片和枝条分别出现典型病斑,接种发病组织再分离所得病菌性状、16S rDNA与初分离的一致,完成柯赫氏法则验证;以分离的核桃黑斑病菌总DNA为模板,采用细菌16SrDNA的通用引物,通过PCR扩增到约1.5 kb的片段,克隆到pMD-18T载体。测序结果表明克隆的16S rDNA片段长为1 453个核苷酸,通过GenBank上BLAST分析,表明该病原细菌为Xanthomonas campestris。

Walnut blight is a severe disease on walnut production.In 2008,a suspected blight disease was found in walnut trees planted in rural area of Beijing.In order to confirm the taxonomic status of the pathogen,bacteria were isolated and purified from plant samples with typical symptoms.Pathogenicity was confirmed by artificial inoculation of healthy walnut branches and leaves with a suspension of an isolated bacterial strain.A 1.5 kb of 16S rDNA fragment amplified by PCR using bacterial 16S rDNA universal primers was cloned into pMD-18T vector.Sequencing analysis revealed that the fragment contains 1453 nucleotides which is highly similar to the 16S rDNA of bacterial pathogen Xanthomonas campestris.